[unreadable] With a few exceptions, the metabolism and possible toxicity of D-amino acids for bacteria is not well understood. We will investigate an array of potentially directly selectable resistance markers to different D-amino acids for use in genetic manipulation of select agent exempt strains of the three NIAID class A agents, Yersinia pestis, Francisella tularensis and Bacillus anthracis. In order to simultaneously detoxify and use D-serine as a carbon and nitrogen source in the urinary tract, uropathogenic strains of Escherichia coli possess the D-serine deaminase locus, dsdCXA. We have intensively studied this system and propose to introduce recombinant versions of the dsdXA genes or homologs into the different class A biodefense agents in order to assess the utility of D-serine deaminase (DsdA) as a non-antibiotic, directly selectable marker. Our preliminary results indicate that Y. pestis is sensitive to D-serine when grown in complex media. [unreadable] We will also assess if D-tyrosine, D-cysteine, D-methionine, D-phenylalanine and D-tryptophan will be inhibit growth of the different agents in complex media and modified versions of their respective defined media. There are a variety of known dissimilatory mechanisms in E. coli and Bacillus subtilus for these particular inhibitory amino acids. We will investigate the ability of known dissimilatory genes to provide a directly selectable phenotype. As an alternative approach, we will assess if recombinant versions of fungal or mammalian derived D-amino acid oxidase genes can be used for direct selection of resistance to single D-amino acids or cocktails of D-amino acids. [unreadable] Our search for directly selectable markers has also led to an examination of tellurite resistance. There are at least five different bacterial gene systems identified that confer such resistance. Our preliminary results indicate that B. anthracis and Y. pestis are sensitive to potassium tellurite in complex media. We will attempt to engineer tellurite resistance markers into the three species. [unreadable] Our exploration of sensitivity and resistance mechanisms for D-amino acids and tellurite in this [unreadable] R21 project is expected to provide multiple avenues for generation of new selective markers in these three agents. Our work is very likely to provide insights into how to engineer similar traits in additional bacterial pathogens of biodefense interest. [unreadable] [unreadable] [unreadable]